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Immunophenotyping (Haematology)

The laboratory is equipped with 2 Beckman Coulter Navios flow cytometers with 10 colour capability. These allow the identification of antibody-antigen reactions by identifying light emitted by fluorochromes conjugated to the antibodies used. The pattern of positive and negative reactions provide the Immunophenotype.

We offer an extensive repertoire of investigations which provide evidence for the diagnosis and classification of haematological disorders. We also offer stem cell quantitation for patients undergoing stem cell harvests, PNH diagnosis  and  quantitation of feto-maternal haemorrhage. Further information may be found below.


Sample requirement:

Immunophenotyping requires fresh blood or bone marrow drawn into EDTA tubes. Marrow should be first pull. Quality of sample is essential to achieve meaningful results.

Please refer to the specific investigation types below, as the different sample types are not always acceptable. Recommended minimum sample for peripheral blood is 3mls. There is no minimum set for bone marrow. We can also investigate fluid samples such as CSF, but these should reach the lab within 4 hours as the cells degrade quickly in these sample types. Fluids may be taken into EDTA or plain universals.

Please note that the number of leukocytes present may impact on our ability to provide full analysis.

Please telephone if you require advice on 0121 424 0704.


Laboratory opening:

The laboratory is open Monday - Friday, 08:30 - 17:00. Latest time of sample receipt for guaranteed same day analysis is 14.00. Samples arriving later than this may be refrigerated until the next working day. Analysis may be performed following weekend storage providing cell viability is within acceptable limits.

Samples should be transported to the laboratory as soon as possible after collection following the transport procedure.  For external requests, samples should be transported in suitable containers via courier and labelled for the attention of Cell Markers, Haematology BHH. Samples should not be sent in the post.



For enquiries telephone 0121 424 0704 to speak with a Biomedical Scientist in the Immunophenotyping Laboratory. For clinical advice please contact the on-call Consultant Haematologist via switchboard 0121 424 2000.


Request forms:

Click here to download a request form (pdf) for haematology investigations, or here to download a request form for feto-maternal haemorrhage.

Please avoid using photocopies of old versions of request forms as these may not be UKAS compliant and may not represent the current tests on offer.

Please complete the request form accurately providing as much relevant information as possible. The laboratory will not accept samples/requests with missing or mislabelled PID.


Sample acceptance criteria:

There must be a minimum of 3 identifiers which link the sample and the request form and should include the patient’s first name, patient’s surname and either hospital number, date of birth or NHS number..


Click for Test Database




The lymphocyte screen is designed to screen for lymphoproliferative disorders. If an abnormal population is identified, an extended panel will be performed to allow characterisation of the cells and provide evidence for diagnosis. Screens which do not identify an abnormality will be reviewed with morphology by a member of the consultant team prior to reporting.



This panel aims to identify numbers of blasts and monocytoid cells. Blasts are identified as myeloid and abnormal phenotypic expression may indicate dysplasia. However, this is not a diagnostic panel and is designed to monitor blast levels. Additional investigations will be performed as necessary if an abnormal population is identified.

When requesting an MDS panel, please note that peripheral blood is unsuitable for diagnostic purposes.

The panel also provides evidence for acute leukaemia by providing a blast percentage. 

All results will be reviewed together with morphology by one of the consultant team prior to reporting.




We have specifically developed panels to give evidence for the diagnosis and classification of haematological malignancies:



ACUTE LEUKAEMIA PANEL for all lineages of acute leukaemias

MYELOMA/MGUS PANEL for the identification of neoplastic plasma cells

Relevant panels will be performed as appropriate following initial screening, or as indicated on the request form. The results will indicate approximate percentage of cells and their phenotypic expression. Please note that plasma cells in particular are fragile and easily lost during sample preparation and staining, and thus percentage may be lower than that seen morphologically.

In addition to diagnostic evidence, the results may provide evidence of prognosis.

All results will be reviewed together with morphology by one of the consultant team prior to reporting.

All new diagnosis acute leukaemias will be telephoned through to the requesting consultant –please provide contact details on the request form.





We use a combination of antibodies to identify CLL cells and differentiate them from other B cells. This results in a sensitive measure of CLL MRD and is used to monitor patient’s response to therapy. 

Reporting of CLL MRD has been updated to provide additional information and will include:

A summary statement

A quantitation (%)                           ONLY if the level is at or above the limit of quantitation

Limit of detection (LOD)             The percentage above which CLL cells can be recognised but there are too few to be reliably quantified.

Limit of quantitation (LOQ)       The minimum percentage at which CLL cells may be quantified.

LOD and LOQ are calculated for each patient sample.

Ability to resolve CLL cells is compromised in patients with an atypical phenotype, and patients on some targeted therapies such as rituximab.


Myeloma MRD:

This utilises the same combination of antisera used in the diagnostic panel, but sensitivity is increased by increasing the number of cells counted.


Acute Leukaemia MRD:

There are specific panels for the recognition, characterisation and quantitation of residual disease in acute lymphoblastic leukaemias.

The approach to MRD in acute myeloid leukaemia is to provide a quantitation of myeloid blasts, wherever possible with reference to leukaemia associated antigens.

MRD levels in acute leukaemias by flow are for guidance only and must be viewed alongside other evidence such as cytogenetics.

All results will be reviewed together with morphology by one of the consultant team prior to reporting.

Residual disease in acute leukaemia

AML – a myeloid blast count will be provided alongside morphological interpretation.

B-ALL and T-ALL – Analysis will be performed with reference to the diagnostic phenotype wherever available.

Consultant review will include morphological inspection.

The sensitivity and specificity of these panels cannot be verified and analysis is for guidance only whilst awaiting other evidence such as cytogenetics.

Please note that paucicellular samples such as CSF are particularly challenging and results are often unhelpful.



Paroxysmal Nocturnal Haemoglobinuria (PNH) is an acquired haematopoietic stem cell disorder resulting in a partial or total deficiency of proteins normally linked to cell membranes by a glycosylphosphatidylinositol (GPI) anchor. Classical features are:

intravascular haemolysis

bone marrow failure

thrombotic tendency

and may also include abdominal pain, dysphagia, erectile dysfunction, renal failure, lethargy. It can often go undiagnosed for months or even years. Flow cytometry can provide a definitive diagnosis.


PNH screening can only be performed on peripheral blood (taken into EDTA). Marrow is unsuitable due to the presence of immature cells.

Investigation for PNH involves the analysis of antigens normally linked via the GPI anchor on red cells, neutrophils and monocytes. Additionally, neutrophil and monocyte screening includes FLAER (FLuorescent AERolysin) which binds directly to GPI. Red cell analysis provides classification into PNH types I and II but clone size may not be representative due to transfusion and/or haemolysis. Neutrophil and/or monocyte analysis provides an accurate measure of clone size and is used to monitor the disorder over time.

Please indicate recent transfusion history on the request form. See Test Database for information on sensitivity.


Click here for further guidance on PNH testing




FMH  by flow cytometry identifies and enumerates the bleed of foetal cells into maternal circulation following birth or other traumatic event. Please note that this screen is based on the principle that maternal samples are Rh D negative and baby/cord samples are Rh D positive. In ante-natal patients or patients who have had an IUD it may not be possible to identify a bleed if mother and baby have the same Rh D type. Also as the principle is based on staining using a fluorescent anti-D, patients who have received recent prophylactic anti-D may give reduced values due to antigen blocking by prophylactic anti-D.

To ensure the correct dose of anti-D is administered we report a suggested dosage taking into account the uncertainty of measurement of FMH by flow in our laboratory. The reported bleed (in mls) is the value obtained by flow cytometry. The suggested dosage of anti-D is based on the reported bleed plus the uncertainty of measurement. 

Current guidelines state that FMH quantitation by flow cytometry should be performed on any bleed greater than 2mls as identified by the Kleihauer stain (performed in the Blood Bank). Patients from HEFT will automatically be referred by the blood bank.


To download the FMH request form click here


For any further advice please telephone the laboratory on 0121 424 0704  

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Bone Marrow Aspirate

All Bone Marrow aspirates will be processed for immunophenotyping – please supply relevant diagnostic information and tick investigation(s) required. If no investigations are indicated screening will proceed based on diagnostic information provided.

Please indicate on the request form if immunophenotyping or molecular studies are required by ticking the relevant boxes. Aspirates should be taken into EDTA and transported to the laboratory as soon as possible where slides will be made and processed for morphological examination and reporting by the consultant led team. In addition to routine Romanowsky stains, iron stores will be assessed via the Perl’s Prussian Blue stain. 

Trephine samples should be directed to the Histopathology Department with an accompanying Histopathology request form.


The Bone Marrow Aspirate Request Form (pdf) can be downloaded from here.

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The laboratories at Heartlands Hospital, Good Hope Hospital and Solihull Hospital form part of the services provided by University Hospitals Birmingham and are UKAS (United Kingdom Accreditation Service) accredited to the ISO 15189:2012 standard. For a list of accredited tests and other information please visit the UKAS website using the following link: https://www.ukas.com/find-an-organisation/

  • Heartlands, Good Hope and Solihull Hospital pathology laboratories are a UKAS accredited medical laboratory No.8217
  • United Kingdom Health Security Agency laboratory is a UKAS accredited medical laboratory No.8213

Tests not appearing on the UKAS Schedule of Accreditation currently remain outside of our scope of accreditation. However, these tests have been validated to the same high standard as accredited tests and are performed by the same trained and competent staff.

For further test information, please visit the test database: http://www.heftpathology.com/frontpage/test-database.html.

Protection of personal information - Laboratory Medicine comply with the Trust Data Protection policy and have procedures in place to allow the Directorate and its employees to comply with the Data Protection act  1998 and associated best practice and guidance.

For further information contact Louise Fallon, Quality Manager, 0121 424 1235